Bile acids, which are synthesized by the liver and secreted by the gallbladder, can be classified into free or conjugated, as well as primary or secondary bile acids [1]. Bile acids are derived from cholesterol through two synthetic pathways via CYP enzymes, resulting in the formation of primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA) [2,3]. These primary bile acids are further conjugated with glycine or taurine in the liver, forming conjugated bile acids, which are stored in the gallbladder and excreted into the intestine with bile. Through the action of intestinal microbiota, conjugated bile acids are hydrolyzed, regenerating free bile acids that undergo oxidation–reduction reactions to produce secondary bile acids, such as deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA) [4]. Most bile acids are reabsorbed into the liver and undergo enterohepatic circulation, resulting in a bile acid pool containing a variety of structurally different bile acids. Over the years, numerous bile acids have been identified. In addition to the bile acids mentioned above, multiple isomers resulting from variations in substitution site and configuration have been found in body fluids and tissues [5]. The structural diversity of bile acids contributes to variations in their hydrophobicity, which subsequently affects their therapeutic or toxic effects [6]. Recent researches have revealed a strong association between bile acids and physiological changes in metabolic disorders, including glucose and lipid metabolism, as well as energy homeostasis [7,8]. Specific bile acids have been implicated in the development and treatment of diseases such as obesity, non-alcoholic fatty liver disease (NAFLD), and type 2 diabetes (T2DM) [9,10]. Especially, some bile acids such as hyocholic acid (HCA) and glycoursodeoxycholic acid (GUDCA) could serve as diagnostic markers for NAFLD, or as indicators for monitoring the progression and remission of diabetes [11], [12], [13], [14], [15], [16]. In addition, CDCA have been shown to be associated with T2DM and can serve as a marker for predicting diabetes remission after gastric bypass surgery [17]. More notably, several bile acids themselves have demonstrated promising effects in reducing glucose and lipid levels [18], [19], [20], [21]. Taurochenodeoxycholic acid (TCDCA), tauroursodeoxycholic acid (TUDCA), and GUDCA have been found to be closely related to the hypoglycemic and lipid-lowering effects of drugs or food [16,22]. underscoring their considerable value in the diagnosis and treatment of metabolic diseases.

Due to the significant physiological and pathological importance, qualitative and quantitative research on bile acids is crucial. A large number of analytical methods have been established so far. However, most studies have focused on a limited number of bile acids [23], [24], [25], [26], [27]. Although some methods determined dozens of bile acids, either the separation of some isomers or the sensitivity was sacrificed [28], [29], [30]. Previous studies have shown that current research on bile acids has gone deep into those with trace amount in human, such as UDCA and its amino acid conjugates, or HCA species mainly derived from pig bile [11,15,31], which brings higher requirements for the quantitative methods. However, quantification of multiple bile acid isomers remains a serious challenge. Increasing the number of bile acid isomers poses challenges to chromatographic separation, especially in terms of balancing the run time and separation capacity. There are still some barriers in the field of multiple bile acid analysis. Firstly, numerous isomers with similar chemical structures and polarity have been found in the previous qualitative and quantitative research [5], which poses a great challenge for their separation in column. Secondly, ionization and fragmentation of bile acids is difficult in MS, making the detection of trace amounts of bile acids hard. More importantly, significant inter-individual variability of bile acids concentrations had been noted in previous studies. This necessitates a broad linear range to achieve accurate quantification of different individual samples as much as possible.

Against this backdrop, the present study endeavors to establish a robust method for the simultaneous quantitative of forty-five bile acids in plasma samples. Using the isomers-oriented separation strategy, most of the bile acids could be well separated and accurately quantified, which has advantage over previous reported methods. Additionally, the validation results demonstrated that the method was sensitive, accurate, and robust for determination of the corresponding bile acids concentrations in human plasma. The dynamic bile acids profile was plotted with the data obtained from current method and the results indicated the possible correlation among glucose, insulin and bile acids, which may provide important information for the determination of biomarkers related to metabolic diseases.